Inhibitors for the blood-clotting factor Xa

ABSTRACT

The invention relates to derivatives of amidino-benzylamine, especially derivatives of 4-amidino-benzylamine, with two bonded amino acids. These derivatives represent a novel group of highly active and very selective F Xa-inhibitors for treating cardiovascular diseases and thrombotic events.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is the National Stage of International Application No.PCT/EP01/06814, filed on Jun. 15, 2001, which claims benefit of DE10029015.9, filed on Jun. 15, 2000.

The invention relates to novel inhibitors for the coagulation factor Xafor the treatment of cardiovascular diseases and for the prevention ofthromboembolic events.

The anticoagulants of the heparin type or the vitamin K antagonistspresently employed clinically do not comply with all requirements for an“ideal” antithrombotic. Therefore alternatives are sought with lowmolecular weight inhibitors of the coagulation enzymes, especially ofthrombin and factor Xa (F Xa). A particular advantage of F Xa inhibitorsin comparison with thrombin inhibitors could be the lower tendency tobleeding which has been shown in various animal experiments. Thus thebleeding time was only minimally influenced in antithromboticallyeffective doses (J. M. Herbert et al., J. Pharmacol. Exp. Ther. 276,1030-1038, 1996; K. Sato et al., Brit. J. Pharmacol. 123, 92-96, 1998).

The first nonpeptide compounds having a high affinity for F Xa weresymmetrical bisbenzamidines (K_(i)=13 nM for the most active compoundBABCH) (J. Stürzebecher et al., Thromb. Res. 54, 245-252, 1998). Thenaphthamidine derivative DX-9065a has two basic groups and inhibits F Xaselectively with K_(i)=24 nM (T. Hara et al., Thromb. Haemost. 71,314-319, 1994). The inhibitor YM-60828 (K. Sato et al., Eur. J.Pharmacol. 339, 141-146, 1997), which is structurally related toDX-9065a, is even more active (K_(i)=1.3 nM). In the meantime, a wholeseries of further bis-basic compounds have been described in which, forexample, two benzamidine residues are linked via an oxazoline ring(K_(i)=18 nM) (M. L. Quan et al., Bioorg. Med. Chem. Lett. 7, 2813-2818,1997) or a carboxymethyl-alkyl chain (K_(i)=34 nM) (T. P. Maduskuie etal., J. Med. Chem. 41, 53-62, 1998). The disadvantage of the bis-basiccompounds is, in particular, the low bioavailability after oraladministration.

Inhibitors for F Xa which only contain one basic group have also beendescribed. N-substituted amidino-phenoxypyridines (K_(i)=0.11 nM forBX-807834) were developed on the basis of BABCH (R. Mohan et al.,Bioorg. Med. Chem. Lett. 8, 1877-1882, 1998; G. B. Phillips et al., J.Med. Chem. 41, 3557-3562, 1998). Amides ofNα-adamantyloxycarbonyl-3-amidinophenyl-alanine (K_(i)=74 nM for themost active compound) are selective inhibitors of F Xa (S. Sperl et al.,Biol. Chem. 381, 321-329, 2000), while Nα-arylsulfonyl-aminoacylatedesters of 3-amidinophenylalanine have a low inhibitory action (K_(i)≈840nM for TAPAM) (J. Stürzebecher et al., Thromb. Res. 54, 245-252, 1998).WO 96/10022 discloses inhibitors which no longer have any strong chargeat all (K_(i)=3.0 nM for the most active compound).

Until now, only a few peptides which are derived from the substratesequence Ile-Glu-Gly-Arg have been described as inhibitors of F Xa. Thechloromethyl ketones described by Kettner and Shaw (Thromb. Res. 22,645-652, 1981) inhibit F Xa irreversibly and are not suitable for invivo applications. On the other hand, the peptides SEL 2489 (K_(i)=25nM) and SEL 2711 (K_(i)=3 nM) are extremely active (J. A. Ostrem et al.,Biochemistry 37, 1053-1059, 1998). Some peptidyl arginine aldehydes havealso been described which, in addition to argininal in the P1 position,have a D-arginine or an unnatural basic amino acid in P3 (Z. H.Jonathan, Bioorg. Med. Lett. 9, 3459-3464, 1999). However, so far nopeptidyl agmatine derivatives are known as inhibitors of F Xa, althoughthis type of inhibitor has led to considerable advances in the furtherdevelopment of thrombin inhibitors. In this case, the successes withcompounds of the D-Phe-Pro-Arg type having a C-terminal agmatine andderivatives derived therefrom were particularly noteworthy. PicomolarK_(i) values were achieved for thrombin inhibition and the oralbioavailability was improved (T. J. Tucker et al., J. Med. Chem. 40,1565-1569 and 3687-3693, 1997). In this case, however, no inhibition ofF Xa was observed. For instance, melagatran, which has a4-amidinobenzylamine residue at the C terminus and is very unspecific,inhibits F Xa with a K_(i)=2.8 μM. On the other hand, trypsin (K_(i)=4.0nM) and thrombin (K_(i)=2.0 nM) are inhibited more than three orders ofmagnitude more strongly (D. Gustafsson et al., Blood Coagul.Fibrinolysis 7, 69-79, 1996).

The invention is based on the object of specifying an active compoundwhich is also suitable for therapeutic applications and inhibits thecoagulation factor Xa with high activity and specificity and which canbe prepared with the lowest possible synthesis expenditure.

Surprisingly, it has been found that acylated amidinobenzylamineaccording to the general formula I shown in Patent claim 1, inparticular compounds of 4-amidinobenzylamine in which X, R₁, R₂ and R₃result in natural and/or unnatural amino acids, inactivate factor Xavery efficaciously and selectively and effectively inhibit thecoagulation of human blood plasma. Amidinobenzylamine in this case formsa particularly active inhibitor of factor Xa if the amidino group is inthe 4-position, glycine and D-serine tert-butyl ether are bonded asamino acids and if the compound has an N-terminal protective group R₄composed of an aryl- or aralkylsulfonyl residue.

Besides factor Xa, other enzymes were markedly less inhibited by theglycine derivatives, such that the derivatives of amidinobenzylamineaccording to the invention are a novel group of highly active and veryselective F Xa inhibitors. In contrast to this, compounds which carry noH as R₁ (e.g. alanine derivatives) no longer selectively inhibit factorXa, but are also strong inhibitors of trypsin, thrombin and plasmin.

The compounds are as a rule present as salts with mineral acids,preferably as hydrochlorides, or as salts with suitable organic acids.

The compounds of the general formula I can be prepared in a manner knownin principle, as described below:

The starting compound 4-cyanobenzylamine is prepared from 4-cyanobenzylbromide via Gabriel synthesis (G. Wagner and I. Wunderlich, Pharmazie32, 76-77, 1977; B. C. Bookser and T. C. Bruice, J. Am. Chem. Soc. 113,4208-4218, 1991). The Boc-protected acetyloxamidino-benzylamine isobtained from the 4-cyanobenzylamine thus prepared. The coupling of thefurther amino acids and of the protective group R₄ is carried out bymeans of standard coupling methods using Boc as an N-terminal protectivegroup. The second amino acid can also be coupled directly as an N-aryl-or N-aralkylsulfonyl-protected amino acid. The peptide analogs aresynthesized sequentially, beginning from the acetyloxamidinobenzylamine.Most of the products crystallize well and can thus be simply purified.The purification of the inhibitors is carried out in the last stage bymeans of preparative, reversed-phase HPLC.

The invention will be illustrated in greater detail below with the aidof three working examples:

WORKING EXAMPLE 1

Synthesis of benzylsulfonyl-D-Ser(Bz)-Gly-4-amidino-benzylamide×HCl

1.1 Boc-4-cyanobenzylamide

20 g (0.151 mol) of 4-cyanobenzylamine were dissolved in 300 ml of H₂O,150 ml of dioxane and 150 ml of 1 N NaOH. 37.5 ml of di-tert-butyldicarbonate were added dropwise with ice cooling and the mixture wasstirred at 0° C. for one hour and at room temperature for a further 24hrs. The dioxane was removed i.v. and the aqueous residue was extracted3 times with ethyl acetate. The combined extracts were washed 3 timeswith 5% strength KHSO₄ solution and 3 times with saturated NaClsolution, dried over Na₂SO₄ and concentrated i.v. (white crystals).HPLC: acetonitrile/H₂O, elution in 44.1% acetonitrile; yield: 30.48 g(0.131 mol), 87%.

1.2 Boc-4-acetyloxamidinobenzylamide

According to Judkins et al. (Synthetic Comm. 26, 4351-4367, 1996), 30.48g (0.131 mol) of Boc-4-cyanobenzylamide were dissolved in 300 ml of abs.ethanol with 13.65 g (0.197 mol) of hydroxylamine×HCl and 34 ml (0.197mol) of DIEA. The solution was refluxed for 2 hrs and stirred at roomtemperature overnight. The batch was then concentrated i.v., the residuewas dissolved in about 200 ml of acetic acid and the solution wastreated with 18.67 ml (0.197 mol) of acetic anhydride. After 1 hr, itwas again concentrated, dissolved in ethyl acetate and washed 3 timeseach with 5% strength KHSO₄ solution and saturated NaCl solution at 0°C. After drying over Na₂SO₄ and concentrating i.v., a white powderprecipitated. HPLC: acetonitrile/H₂O, elution in 32.0% acetonitrile;yield: 31.3 g (0.102 mol) 78%.

1.3 4-acetyloxamidinobenzylamine×HCl

5 mmol of Boc-4-acetyloxamidinobenzylamide are dissolved in 20 ml of 1 NHCl in glacial acetic acid and the solution is allowed to stand at roomtemperature for 45 min. It is then largely concentrated i.v., and theproduct is precipitated using dry diethyl ether, filtered off on a frit,washed again with fresh ether and dried i.v. On account of thequantitative reaction, the product was employed for the next synthesisstep without further purification.

1.4 Boc-Gly-4-acetyloxamidinobenzylamide

The coupling of Boc-Gly-OH (Orpegen, Heidelberg) to4-acetyloxamidinobenzylamine was carried out according to Frérot et al.(Tetrahedron 47, 259 ff., 1991). To this end, 2.064 g (9.3 mmol) of4-acetyloxamidinobenzylamine×HCl and 1.629 g (9.3 mmol) of Boc-Gly-OHwere dissolved in about 25 ml of DMF. 4.84 g (9.3 mmol) of PyBOP and3.878 ml (27.9 mmol) of TEA were then added at 0° C. and the pH wasadjusted to 9 using TEA. After stirring at room temperature for 1 hr,the mixture was concentrated i.v., taken up in ethyl acetate andsubjected to acidic, basic and neutral washing 3 times each, dried andconcentrated. Yield: 3 g (8.2 mmol) 88%.

1.5 Boc-Gly-4-amidinobenzylamide×AcOH

3 g (8.2 mmol) of Boc-Gly-4-Acetyloxamidinobenzylamide were dissolved in200 ml of 90% strength acetic acid. 300 mg of 10% palladium on activatedcarbon were then added under argon. Argon was replaced by a hydrogenatmosphere and the batch was hydrogenated for 24 hrs with vigorousstirring. The catalyst was filtered off and the filtrate wasconcentrated i.v. Yield: 2.9 g (7.9 mmol) 96%.

1.6 H-Gly-4-amidinobenzylamide×2 HCl

2.9 g (7.9 mmol) of Boc-Gly-4-amidinobenzylamide were dissolved in 100ml of 1 N HCl in glacial acetic acid and the solution was allowed tostand at room temperature for 45 min. It was then largely concentratedi.v. and precipitated using dry diethyl ether, then filtered off on afrit and the product was again washed with fresh ether. After drying theproduct i.v., it was used without further purification for the synthesisaccording to item 1.8.

1.7 Benzylsulfonyl-D-Ser(Bz)-OH

229 mg (1.173 mmol) of H-D-Ser(Bz)-OH and 408 μl (2.345 mmol) of DIEAwere dissolved in 50 ml of 50% acetonitrile. 335 mg (1.76 mmol) ofbenzylsulfonyl chloride were then added and the mixture was stirred atroom temperature for 12 hrs. It was concentrated i.v., taken up usingethyl acetate and subjected to acidic and neutral washing 3 times each.After drying over sodium sulfate, it was concentrated i.v. Yield: 289 mg(0.827 mmol) 71%.

1.8 Benzylsulfonyl-D-Ser(Bz)-Gly-4-amidinobenzylamide×TFA

151 mg (0.433 mmol) of benzylsulfonyl-D-Ser(Bz)-OH and 121 mg (0.433mmol) of H-Gly-4-amidinobenzylamide×2 HCl were dissolved in a littleabs. DMF. 225 mg (0.433 mmol) of PyBOP and 230 μl (1.32 mmol) of DIEAwere added with ice cooling. After stirring at room temperature for 1hr, the mixture was concentrated i.v. and the product was purified bymeans of HPLC (acetonitrile/H₂O, 0.1% trifluoroacetic acid, elution in37.4% acetonitrile). Yield: 232 mg (0.356 mmol) 82%.

WORKING EXAMPLE 2

Inhibition of F Xa by selected compounds having Y = amidino R₃ con-figura- Amidino R₄ tion R₃ R₂ X-R₁ position K_(b) μM H L CH₂—OH H CH₂4 >1000 Boc L CH₂—OH H CH₂ 4 110 H D CH₂—OH H CH₂ 4 >1000 Ac D CH₂—OH HCH₂ 4 >1000 Bz—SO₂ D CH₂—OH H CH₂ 4 3.0 Bz—SO₂ D CH₂—O— H CH₂ 4 0.050 BzBz—SO₂ D CH₂—O— H CH₂ 4 0.030 tBu Bz—SO₂ D CH₂—O— H CH₂— 4 0.044 tBu CH₃H D CH₂—O— H CH₂ 3 140 Bz Boc D CH₂—O— H CH₂ 3 93 Bz Bz—SO₂ D CH₂—O— HCH₂ 3 84 BzDetermination of the Inhibitory Action

For the determination of the inhibitory action, 200 μl of tris buffer(0.05 M, 0.154 M NaCl, 5% ethanol, pH 8.0; contains the inhibitor), 25μl of substrate (Moc-D-Nle-Gly-Arg-pNA in H₂O; Pentapharm Ltd., Basle,Switzerland) and 50 μl of F Xa (from Rind, Diagnostic Reagents Ltd,Thame, GB) were incubated at 25° C. After 3 min, the reaction wasinterrupted by addition of 25 μl of acetic acid (50%) and the absorptionat 405 nm was determined by means of microplate reader (MR 5000,Dynatech, Denkendorf, Germany). The K_(i) values were determinedaccording to Dixon (Biochem. J. 55, 170-171, 1953) by linear regressionby means of a computer program. The K_(i) values are the mean of atleast three determinations.

WORKING EXAMPLE 3

Inhibition of the coagulation of human plasma bybenzylsulfonyl-D-Ser(Bz)-Gly-4-amidinobenzylamide Prolongation of thecoagulation time Concentration (%) μM aPTT PT 3.3 385 386 1.7 260 2660.83 185 198 0.42 146 153 0.21 122 127 0.1 111 119Determination of the Inhibition of Coagulation

For the determination of the prothrombin time (PT), 100 μl ofthromboplastin (Dade, Unterschleiβheim) and 100 μl of inhibitor,dissolved in CaCl₂ (0.05 M, 5% ethanol) were incubated at 37° C. for 2min and the coagulation was started by addition of 100 μl of humancitrate plasma. For the determination of the activated partialthromboplastin time (aPTT), 100 μl of human citrate plasma wereincubated with 100 μl of aPTT reagent (Roche Diagnostics, Mannheim) at37° C. for 3 min and the coagulation was started by addition of 100 μlof inhibitor, dissolved in CaCl₂ (0.05 M, 5% ethanol). The coagulationtimes were determined using the Thrombotrack coagulometer (Immuno,Heidelberg).

Abbreviations used: Ac acetyl Boc tert-butyloxycarbonyl Bz benzyl DIEAdiisopropylethylamine DMF N,N-dimethylformamide i.v. in vacuo PyBOPbenzotriazol-l-yl-N-oxytris- (pyrrolidino) phosphoniumhexafluorophosphate TEA triethylamine TFA trifluoroacetic acid THFtetrahydrofuran tBu tert-butyl

1. Inhibitors for the coagulation factor Xa of the formula:

in which the substituent Y occurs in the 3- or 4-position and is anamidino group

in which R₅ is selected from the group consisting of H, OH, —CO—R₇ and—COO—R₇, where R₇ is selected from a branched or unbranched alkyl having1-16 C atoms, a substituted or unsubstituted aryl or heteroaryl groupand a substituted or unsubstituted aralkyl or heteroaralkyl group, X isCH or N, R₁ is H or a branched or unbranched alkyl having 1-8 C atoms or(CH₂)_(n)—OH where n=1-5, R₂ is H or a branched or unbranched alkylhaving 1-8 C atoms, R₃ is a branched or unbranched alkyl having 1-8 Catoms or (CH₂)_(n)—O—R₆, where N=1-5 and R₆ is a branched or unbranchedalkyl having 1-6 C atoms, a substituted or unsubstituted aryl orheteroaryl group or a substituted or an unsubstituted aralkyl orheteroaralkyl group and R₄ is selected from the group consisting of—SO₂—R₈, —CO—R₈, —COO—R₈ and H, where R₈ is a branched or unbranchedalkyl having 1-16 C atoms, a substituted or an unsubstituted aryl orheteroaryl group, a substituted or unsubstituted aralkyl orheteroaralkyl group, an adamantyl group, a camphoryl group or acyclohexylmethyl group.
 2. Inhibitors according to claim 1, wherein Y isin the 4-position, X is CH, R₁ and R₂ are H, R₃ is (CH₂)_(n)—O—R₆, wheren is 1 and R₆ is t-butyl, wherein R₃ is in the D configuration, and R₄is —SO₂—R₈, wherein R₈ is a substituted or unsubstituted aryl or aralkylgroup.
 3. A method of preventing or treating thromboembolic diseasescomprising administering to a patient a factor Xa inhibitor of claim 1.4. The method of claim 3, wherein the factor Xa inhibitor is in the formof a tablet, coated tablet, capsule, pellet, suppository, solution orpatch.
 5. Inhibitors for the coagulation factor Xa of the formula:

in which the substituent Y occurs in the 3- or 4-position and is anamidino group

in which R₅ is selected from the group consisting of H, OH, —CO—R₇ and—COO—R₇, where R₇ is selected from a branched or unbranched alkyl having1-16 C atoms, a substituted or unsubstituted aryl or heteroaryl groupand a substituted or unsubstituted aralkyl or heteroaralkyl group, X isCH or N, R₁ is H or a branched or unbranched alkyl having 1-8 C atoms or(CH₂)_(n)—OH where n=1-5, R₂ is H or a branched or unbranched alkylhaving 1-8 C atoms, R₃ is (CH₂)_(n)—S—R₆ or (CH₂)_(n)—NH—R₆ where n=1-5and R₆ is a branched or unbranched alkyl having 1-16 C atoms, asubstituted or unsubstituted aryl or heteroaryl group or a substitutedor an unsubstituted aralkyl or heteroaralkyl group and R₄ is selectedfrom the group consisting of —SO₂—R₈, —CO—R₈, —COO—R₈ and H, where R₈ isa branched or unbranched alkyl having 1-16 C atoms, a substituted or anunsubstituted aryl or heteroaryl group, a substituted or unsubstitutedaralkyl or heteroaralkyl group, an adamantyl group, a camphoryl group ora cyclohexylmethyl group.
 6. A method of preventing or treatingthromboembolic diseases comprising administering to a patient a factorXa inhibitor of claim
 5. 7. The method of claim 6, wherein the factor Xainhibitor is in the form of a tablet, coated tablet, capsule, pellet,suppository, solution or patch.